Initiation of HIV-1 reverse transcription occurs at the primer binding site (PBS), which is complementary to the 3' terminal nucleotides of the cellular tRNALys,3. Studies from this laboratory during the last funding period have clearly demonstrated in that mutating the PBS to be complementary to alternative tRNAs resulted in viruses that could utilize these tRNAs to initiate reverse transcription. However, upon limited in vitro culture, all these viruses reverted back to utilize tRNALys,3 to initiate reverse transcription, suggesting that HIV-1 has evolved a specific mechanism to utilize this tRNA. Studies from this laboratory have found that the major determinant for the selection of tRNA used to initiate reverse transcription are found within the U5 and PBS regions of the viral RNA genomes. Using this information, we have developed and characterized unique viruses which utilize alternative tRNAs for initiation of reverse transcription. Furthermore, we have also developed a novel complementation system using an HIV-1 proviral genome modified to be infectious only if co-transfected with in vitro transcribed tRNAPhe. This system now, for the first time, provides us with the opportunity to determine the importance of specific RNA sequences in the primer tRNA as well as the U5 PBS region of the viral RNA genome required for initiation of reverse transcription. The following Specific Aims are proposed: Specific Aim 1: To characterize the selection process for the tRNA used for initiation of reverse transcription. We will utilize the complementation system to characterize the intracellular stability and distribution of the transfected tRNA. Specific Aim 2: To delineate, at the molecular level, interactions between tRNA and U5 PBS required for initiation of reverse transcription. Using mutagenesis, we will create mutant tRNA molecules to be analyzed for the capacity to complement the defective proviral genome that will allow determination of the critical regions of tRNA required for initiation of reverse transcription. Specific Aim 3: To establish that availability of tRNA can influence initiation of reverse transcription and subsequent viral replication. The replication of viruses which stably utilize alternative tRNAs (non-tRNALys,3) for initiation of reverse transcription will be studied in peripheral blood mononuclear cells (PBMC) cultures. Preliminary experiments demonstrate that the replication of these viruses in PBMC is compromised, and we will determine if the defect is due to availability of the tRNA required for initiation of reverse transcription. The long-term goal of these studies will be to delineate the features of both the tRNA as well as the U5 PBS of the viral RNA genome required for selection and positioning of the tRNA at the PBS required for initiation of HIV-1 reverse transcription. The results of these studies will be important for the development of new strategies to inhibit HIV-1 reverse transcription, as well as the further development of HIV-1 as a gene therapy vector.